pdfs.semanticscholar.org · 2018-12-12 · iii acknowledgements as i reach this stage in my life,...

Chemical Constituents from Elytropappus rhinocerotis and Rhoicissus

tridentata: Structural and Activity Studies

By

BONGIWE PRIDESWORTH MSHENGU

Submitted in the fulfilment of the academic requirements for the degree

DOCTOR OF PHILOSOPHY

In the School of Chemistry and Physics

College of Agriculture, Engineering and Science

University of KwaZulu-Natal

Pietermaritzburg

Supervisor: Professor Fanie R. van Heerden

October 2015

ii

Declaration

I hereby certify that this research is a result of my own investigation, which has not

already been accepted in substance for any degree and is not being submitted in

candidature for any other degree.

Signed……………..

Bongiwe Pridesworth Mshengu

I hereby certify that this statement is correct

Singed……………...

Professor Fanie R. van Heerden (Supervisor)

iii

Acknowledgements

As I reach this stage in my life, my heart is filled with joy and great love for my

Almighty God who has always given me the strength to carry on even when I was

walking in the darkest valley. Glory be to the Father, the Son, and the Holy Spirit.

I express my sincere gratitude to my supervisor, Professor Fanie R. van Heerden for her

supervision, guidance, and encouragement throughout my studies.

I have to thank Professor Siegfried E. Drewes, who although not my supervisor has

always been open to discussion and has offered me a lot of guidance throughout my

studies.

My sincere thanks also go to the following people:

Mr. C. Grimmer for his assistance with NMR experiments

Mrs. C. Janse Van Rensburg for helping with mass spectrometry

Dr. C. Southway and Ms P. Lubanyana for assisting with HPLC

Mr. F. Shaik, B. Dlamini, and S. Ball for technical assistance

My special thanks are due to my fellow PhD and MSc colleagues in the Warren

Laboratory, who better understood my everyday struggles in the lab and were always

willing to share constructive ideas with me.

My greatest thanks go to my mother (Mrs. Z. Madlala) and father (Mr. B. Madlala) for

their support and prayers throughout all the years I have been at the university.

I am deeply grateful to my husband (Bonginkosi Mshengu) for his unconditional love

and support that he has given me. My husband had to be the father and mother to our

son (Nkanyiso) while I was preoccupied with my studies and he made sure we had

healthy home-cooked meals now and then. I am deeply thankful to you Donga.

I thank the University of KwaZulu-Natal and National Research Foundation (NRF) of

South Africa for the financial support.

iv

Abstract Traditional medicines are used by approximately 80% of South African population for

their primary health care needs, but the chemistry and biological activity of many

medicinal plants have not yet been investigated. This study focused on the isolation and

structural elucidation of natural products, as well as developing a high-performance

liquid chromatography (HPLC) method to fingerprint the crude extract of Elytropappus

rhinocerotis (L.f.) Less. E. rhinocerotis is well known in traditional medicine for the

treatment of colic, wind, diarrhoea, indigestion, dyspepsia, gastric ulcers and stomach

cancer. This study was also conducted to isolate, elucidate structures and evaluate the

uterotonic activity of natural products from Rhoicissus tridentata (L.f.) Wild & Drumm.

subsp. cuneifolia, a medicinal plant used by many South African women to induce

labour and to tone the uterus during pregnancy.

From the ethyl acetate extract of the aerial parts of E. rhinocerotis, 6,7-

dimethoxycoumarin, 5,6,4'-trihydroxyflavone, 5,7-dihydroxy-4'-methoxyflavone, 5,7-

dihydroxy-6,4'-dimethoxyflavone, kaempferol-3-methyl ether, (+)-13-epi-labdanolic

acid, (+)-labdanolic acid, (+)-labdanolic acid methyl ester, and (+)-labdanediol were

isolated. These compounds are reported for the first time from E. rhinocerotis. The

isolated flavonoids may justify the traditional use of this plant in the treatment of

cancer, while the labdane diterpenes have shown anti-inflammatory activities in other

studies. A HPLC method to fingerprint the crude extract from the aerial parts of E.

rhinocerotis was successfully developed and minor variations were observed in the

chemical compositions of E. rhinocerotis plants collected from different geographic

locations.

From the acetone fraction of the methanol extracts of the root of R. tridentata, catechin,

quercetrin, morin 3-O-α-L-rhamnopyranoside, trans-resveratrol glucoside, an

inseparable mixture of asiatic acid and arjunolic acid, β-sitosterol, and linoleic acid

were isolated and characterised. Except for catechin and β-sitosterol, these compounds

are reported for the first time from Rhoicissus and the occurrence of morin 3-O-α-L-

rhamnopyranoside is reported for the first time from the family Vitaceae.

The uterotonic activity of the crude methanol extract of the root of R. tridentata as well

as the activity of the isolated pure compounds was evaluated using the isolated uterine

v

smooth muscle strips obtained from stilboestrol-primed Sprague-Dawley rats. The

mixture of asiatic acid and arjunolic acid showed a response of approximately 13% in

the force of uterine muscle contractility at 1.23 µg/mL while β-sitosterol demonstrated a

change of 40% in the force of uterine muscle contractility at a concentration of 57.1

µg/mL. Hence it was concluded that the mixture of asiatic acid and arjunolic acid is the

most uteroactive component in the extract of R. tridentata. Morin 3-O-α-L-

rhamnopyranoside and trans-resveratrol glucoside caused a relaxation in the

contractions of the uterine smooth muscle. Both compounds showed a higher inhibition

in the force of contractions when compared to the rate of contractions. These findings

confirmed that R. tridentata possesses both oxytocic and tocolytic activities at different

dosages. Catechin and quercetrin were cytotoxic to the uterine smooth muscle tissue.

vi

Table of Contents Declaration ii

Acknowledgements iii

Abstract iv

List of Figures ix

List of Tables xii

List of Abbreviations xiii

CHAPTER 1: Introduction and aims of study 1

1.1 General introduction 1

1.2 Aims of the study 7

1.3 Organization of the thesis 8

CHAPTER 2: The phytochemistry of Elytropappus rhinocerotis 9

2.1 Introduction 9

2.2 Economic and medicinal uses of some Asteraceae plants 10

2.3 Medicinal uses of some South African Asteraceae plants 15

2.4 The genus Elytropappus 23

2.4.1 Introduction 23

2.4.2 Traditional uses 25

2.4.3 Phytochemistry and biological activities 25

2.5 Results and discussion 27


2.5.2 6,7-Dimethoxycoumarin (2.52) 27

2.5.3 5,7,4'-Trihydroxyflavone (2.53) 30

2.5.4 5,7-Dihydroxy-4'-methoxyflavone (2.54) 32

2.5.5 5,7-Dihydroxy-6,4'-dimethoxyflavone (2.55) 34

2.5.6 Kaempferol 3-methyl ether (2.56) 37

2.5.7 (+)-13-epi-Labdanolic acid (2.57) 39

2.5.8 Synthesis of the p-bromophenacyl ester of (+)-13-epi-labdanolic acid (2.61) 42

2.5.9 (+)-Labdanolic acid (2.62) 45

2.5.10 Synthesis of p-bromophenacyl ester of (+)-labdanolic acid (2.63) 48

vii

2.5.11 (+)-Labdanolic acid methyl ester (2.64) 49

2.5.12 (+)-Labdanediol (2.66) 51

2.5.13 Conclusion 52

2.6 HPLC studies 53


2.6.2 Analysis of E. rhinocerotis collected from location 1 on farm Weltevreded. 54



2.6.5 Conclusion 62

2.7 Experimental 62

2.8.1 Instrumentation and chemicals 62

2.8.2 Plant material 64

2.8.3 Extraction and isolation 64

2.8.4 Physical data of isolated compounds 65

2.8.5 Synthesis of p-bromophenacyl ester of (+)-13-epi-labdanolic acid (2.61) 68

2.8.6 Synthesis of p-bromophenacyl ester of (+)-labdanolic acid (2.63) 69

CHAPTER 3: The phytochemistry of Rhoicissus tridentata 71

3.1 Introduction 71

3.2 Non-South African oxytocic plants 74

3.3 South African oxytocic plants 80

3.3.1 Toxicity of some South African oxytocic plants 89

3.4 Genus Rhoicissus 91


3.4.2 Local uses 93

3.4.3 Biological activities and phytochemistry 94

3.5 Rhoicissus tridentata 96


3.5.2 Biological activities and phytochemistry 97

3.6 Results and discussion: Isolation and structural elucidation of compounds from R. tridentata 101

viii


3.6.2 Quercetrin (3.61) 102

3.6.3 Morin 3-O-α-L-rhamnopyranoside (3.62) 105

3.6.4 Catechin (3.63) 107

3.6.5 Trans-resveratrol glucoside (3.64) 109

3.6.6 Asiatic acid (3.65) and arjunolic acid (3.66) 112

3.6.7 β-Sitosterol (3.22) 115

3.6.8 Linoleic acid (3.18) 117

3.6.1 Conclusion 118

3.7 Results and discussion: Biological activity 119


3.7.2 Uteroactivity assays of the crude R. tridentata extracts 120

3.7.3 Uteroactivity assays of the isolated compounds 123

3.7.4 Uteroactivity assays of Gunnera perpensa 126

3.8 Conclusion 127

3.9 Experimental 128

3.9.1 General experimental procedure 128

3.9.2 Plant material 128

3.9.3 Extraction of the leaves 129

3.9.4 Extraction and isolation of the roots 129

3.9.5 Physical data of the isolated compounds 131

3.9.6 Uteroactivity assays 133

CHAPTER 4: Conclusions 134

References 137

Appendix 1: NMR spectra of isolated compounds 170

Appendix 2: Conference presentations 281

ix

List of Figures

Figure 2.1: Habitat and aerial parts of E. rhinocerotis ................................................. 24

Figure 2.2: UV/Vis absorption spectrum of compound 2.52 ........................................ 28

Figure 2.3: Key HMBC correlations in compound 2.52 .............................................. 29


Figure 2.5: Key HMBC correlations in compound 2.53 .............................................. 31


Figure 2.7: Some HMBC correlations in compound 2.54 ............................................ 33


Figure 2.9: Important HMBC correlations in compound 2.55 ..................................... 36

Figure 2.10: UV/Vis absorption spectrum of compound 2.56 ...................................... 37

Figure 2.11: HMBC correlation in compound 2.56 ..................................................... 38

Figure 2.12: Important HMBC correlations in compound 2.57.................................... 40

Figure 2.13: NOE correlations observed for compound 2.57 ....................................... 40

Figure 2.14: The unit cell of compound 2.61. ............................................................. 43

Figure 2.15: A partially labelled thermal ellipsoid plot of compound 2.61 showing 50% probability surfaces. All hydrogen atoms are shown as small spheres of arbitrary radius. ................................................................................................................................... 43

Figure 2.16: Hydrogen-bonded chains adopted by compound 2.61 running parallel to the b-axis. Hydrogen bonds between the molecules are shown in blue lines and red lines represent all bonds to atoms participating in hydrogen bonding. ................................. 44

Figure 2.17: NOE correlations observed in compound 2.62 ........................................ 46

Figure 2.18: NOE correlations observed for compound 2.64 ....................................... 50

Figure 2.19: HPLC chromatogram and UV/Vis absorption spectra of flavonoids 2.52-2.56 from E. rhinocerotis (location 1). ........................................................................ 55

Figure 2.20: HPLC-PDA chromatogram of E. rhinocerotis collected from location 1 on farm Weltevreded. ...................................................................................................... 56

Figure 2.21: HPLC-LCMS chromatogram of the aerial parts of E. rhinocerotis collected from location 1 on farm Weltevreded. ........................................................................ 56

x

Figure 2.22: HPLC-LCMS chromatogram of the aerial parts of E. rhinocerotis collected from location 1 on farm Weltevreded. ........................................................................ 57

Figure 2.23: HPLC-PDA/LCMS chromatogram of the leaves of E. rhinocerotis collected from location 2 on farm Weltevreded........................................................... 58

Figure 2.24: HPLC-PDA/LCMS chromatogram of the branches of E. rhinocerotis collected from location 2 on farm Weltevreded........................................................... 59

Figure 2.25: HPLC-PDA/LCMS chromatogram of the leaves of E. rhinocerotis collected from location 3 on farm Weltevreded........................................................... 60

Figure 2.26: HPLC-PDA/LCMS chromatogram of the branches of E. rhinocerotis collected from location 3 on farm Weltevreded........................................................... 61

Figure 3.1: Three-dimensional structure of cyclotides kalata B1 (Saether et al., 1995) 78

Figure 3.2: Structures of uterotonic compounds isolated from some oxytocic plants. .. 84

Figure 3.3: Distribution of Rhoicissus. ........................................................................ 92

Figure 3.4: Leaves and branches of R. tridentata ........................................................ 97


Figure 3.6: Key HMBC correlations in compound 3.61 ............................................ 104

Figure 3.7: HMBC correlations in compound 3.62.................................................... 106


Figure 3.9: HMBC correlations in compound 3.63.................................................... 108

Figure 3.10: UV/Vis absorption spectrum of compound 3.64 .................................... 109

Figure 3.11: Important HMBC correlations in compound 3.64.................................. 111

Figure 3.12: HMBC correlations in a partial structure of compound 3.65 and 3.66 ... 113

Figure 3.13: HMBC correlations in a partial structure of compound 3.65 .................. 113

Figure 3.14: HMBC correlations in compound 3.22 .................................................. 116

Figure 3.15: Effects of R. tridentata leaves crude extract on the force and rate of uterine muscle contractility................................................................................................... 120

Figure 3.16: Effects of R. tridentata root crude extract on the force and rate of uterine muscle contractions. ................................................................................................. 121

Figure 3.17: Uterine muscle contractility effects of the acetone fraction from the roots of R. tridentata. ........................................................................................................ 122

xi

Figure 3.18: Uterine muscle contractility effects of the methanol fraction from the roots of R. tridentata. ........................................................................................................ 122

Figure 3.19: Effect of the mixture of asiatic acid and arjunolic acid on the force and rate of uterine muscle contractility. ................................................................................. 124

Figure 3.20: Effect of β-sitosterol on the force and rate of uterine muscle contractility. ................................................................................................................................. 124

Figure 3.21: Effect of morin 3-O-α-L-rhamnopyranoside on the force and rate of uterine muscle contractility. ................................................................................................. 125

Figure 3.22: Effect of trans-resveratrol glucoside on the force and rate of uterine muscle contractility. ............................................................................................................. 126

Figure 3.23: Effect of G. perpensa root aqueous extract on the force and rate of uterine muscle contractility. ................................................................................................. 127

xii

List of Tables

Table 2.1: 1H and 13C NMR data (400 MHz, CDCl3) of compounds 2.57 and 2.62. .... 47

Table 3.1: Some frequently used herbal ingredients of isihlambezo and their active compounds. 82

Table 3.2: 1H and 13C NMR spectroscopic data (CD3OD) of compounds 3.65 and 3.66 ................................................................................................................................. 114

xiii

List of Abbreviations

AcOH : acetic acid

AIDS : acquired immune deficiency syndrome

Å : angstrom

ALT : aspartate transaminase

APCI : atmospheric pressure chemical ionization

BCE : Before the Common Era

CCl4 : carbon tetrachloride

CDCl3 : deuterated chloroform

DM : diabetes mellitus

DCM : dichloromethane

DMF : dimethylformamide

DMSO : dimethyl sulfoxide

ESI : electrospray ionization

EtOAc : ethyl acetate

FDA : Food and Drug Administration

GC-MS gas chromatography-mass spectrometry

Glc : glucose

GPCR : G-protein-coupled receptors

EC50 : half maximal effective concentration

IC50 : half maximal inhibitory concentration

Hex : hexane

HPLC : high-performance liquid chromatography

HRMS : high-resolution mass spectrometry

HIV : human immunodeficiency virus

LPO : lipid peroxidase

xiv

LRMS : low-resolution mass spectrometry

MeOH : methanol

MIC : minimum inhibitory concentration

NP : natural product

NNRTIs: non-nucleoside reverse-transcriptase inhibitors

NMR : nuclear magnetic resonance

NOE : nuclear overhauser effect

NRTIs : nucleoside reverse-transcriptase inhibitors

PDA : photodiode-array detector

PPH : post-partum hemorrhage

PGE2 : prostaglandin E2

PIs : protease inhibitors

RF : retention value

ASP : serum alanine transaminase

TI : therapeutic index

TLC : thin-layer chromatography

TGI : total growth inhibition

TCM : traditional Chinese medicine

UV : ultraviolet

VCD : vibrational circular dichroism

WHO : World Health Organization

CHAPTER 1: Introduction and Aims of Study

1

CHAPTER 1: Introduction and aims of study

1.1 General introduction

Since ancient times, humans have relied on medicinal plants to treat a wide spectrum of

diseases and these plants have formed the basis of various sophisticated traditional

medicine systems (Cragg and Newman, 2013). The Chinese Materia Medica was

amongst the earliest documented systems, with the first record dating to 1100 BCE

(Huang, 2010). The first records on the Indian Ayurvedic system, namely Charaka (341

drugs), Sushruta and Samhita (561 drugs), dates before 1000 BCE (Dev, 2001; Kapoor,

1989). A famous record, known as “Ebers Papyrus”, listing over 700 drugs used in

Egypt, was documented in 1500 BCE (Borchardt, 2002).

Approximately 1000 plant-derived products used in Mesopotamia were documented as

early as 2600 BCE and these products are still used today to treat several diseases.

Examples of these products are the oils from Cedrus species (cedar), Cupressus

sempevirens (cypress), Glycyrrhiza glabra (licorice), Commiphora species (myrrh), and

Papaver somniferum (poppy juice) (Cragg and Newman, 2013). While plants have been

a source of human medicines for thousands of years, the isolation of bioactive

components from plants only started about 200 years ago (Cragg and Newman, 2013).

Some of the early plant-derived drugs discovered were morphine (1.1), which was

isolated from Papaver somniferum L. (Papaveraceae), aspirin (1.3), which is a synthetic

analogue of salicylic acid (1.2) present in willow bark (Salicaceae), quinine (1.4),

isolated from Cinchona species e.g. C. officinalis (Rubiaceae) and digoxin (1.5),

obtained from Digitalis lanata Ehrh. (Plantaginaceae). These drugs, which are still used

today, show activity against pain, rheumatism and headache, malaria, arrhythmia and

congestive heart failure respectively (Buss et al., 2003; Butler, 2004; Rishton, 2008;

Schuster and Wolber, 2010).


2

OH

OH

ONCH3

H

1.1

OAc

COOH

1.3

COOH

OH1.2

1.4N

H3CO

OH N

O O

H

OH

H

H

HOO

OH

OO

OH

OO

OH

OH

1.5

The diverse biological activities exhibited by compounds such as 1.1-1.5 prompted

many laboratories and pharmaceutical companies to embark on natural product (NP)

research. During the 1990’s, 80% of drugs were NP or derived from NP. From the year

2000 to 2002, NP or NP-derived drugs were amongst the 35 top-selling drugs

worldwide (Butler, 2004; Kingston, 2010). Regardless of this great contribution of NP

in drug discovery, most pharmaceutical companies lost interest in NP research during

the period of 2001-2008. Approximately 25% and 50% of marketed drugs at that time

were from natural sources. The loss of interest in NP was attributed to difficulties

experienced in the isolation and identification of hit compounds from crude extracts

(Butler, 2004; Kingston, 2010; Li and Vederas, 2009).

At that stage, combinatorial chemistry was identified as the solution to drug discovery

as it allowed for the fast production of large numbers of compounds as potential drugs.

However, the libraries created through this method often did not produce novel

structural types of compounds. This can be attributed to the difficulty in synthesizing

complex structures to produce novel compounds. As a result, many scientists regained

interest in NP research for drug discovery and development since the complexity of NP

structures makes them good lead compounds. In addition, advances in modern

technology such as molecular modeling, virtual screening, high-throughput cell-based


3

screenings and advanced spectroscopic methods for structural elucidation ensured

quicker identification of hit compounds from the crude plant extracts (Kingston, 2010;

Rishton, 2008; Schuster and Wolber, 2010).

In particular, natural products have contributed to the treatment of infectious,

neurological, cardiovascular and metabolic, immunological and inflammation, and

oncological diseases (Fennell et al., 2004; Kingston, 2010; McGaw et al., 2008; Mishra

and Tiwari, 2011; Mukhtar et al., 2008; Newman and Cragg, 2012). Example of plant-

derived drugs and lead compounds in clinical use for the treatment of infectious

diseases are artemisinin (1.6) and betulinic acid (1.8).

Artemisinin (1.6) is an antimalarial drug isolated from Artemisia annua (sweet

wormwood, qinghao), a plant with long historical use in Traditional Chinese Medicine

(TCM) for the treatment of fevers. This compound was also isolated from several other

Artemisia species (for example, A. vulgaris, A. japonica, A. vulgaris L. (mugwort) syn,

and A. nilagirica), and it is used in many countries for its antimalarial activity (Efferth,

2009; Rashmi et al., 2014). Besides the antimalarial properties, 1.6 and its derivatives

have shown in vitro anti-cancer activity against radiation-resistant breast cancer cells

(Singh and Lai, 2001), drug-resistant small cell lung carcinoma cells (Sadava et al.,

2002), human leukemia cell lines (Lai and Singh, 1995), and colon cancer and active

melanomas (Efferth et al., 2001).

In addition, 1.6 demonstrated antifungal activity against some plant pathogens (for

instance, Gaeumannomyces graminis var. tritici, Rhizoctonia cerealis, Gerlachia nivalis

and Verticillium dahlia) (Tang et al., 2000). A synthetic analogue of artemisinin,

arterolane (1.7) in combination with piperaquine phosphate, is in Phase III clinical trials

for the treatment of malaria in India, Bangladesh, and Thailand (Mishra and Tiwari,

2011).


4

OO

O

H

H

O

1.6

OO

O

O

NHNH2

1.7

Betulinic acid (1.8) was isolated as an anti-HIV principle from the leaves of Syzigium

claviflorum. This compound showed inhibition of HIV-1 replication in H9 lymphocytes

with an EC50 of 1.4 µM and a therapeutic index (TI) of 9.3(Lee, 2010; Mishra and

Tiwari, 2011; Schuster and Wolber, 2010; Sun et al., 1998). Compound 1.8 is present in

many plant species and considerable quantities of this compound can be obtained from

the bark of the birch tree (Betula spp., Betulaceae) (Moghaddam et al., 2012; Pisha et

al., 1995). Betulinic acid was reported to exhibit a variety of other biological properties,

for example, anti-bacterial (Chandramu et al., 2003), anti-malarial (Bringmann et al.,

1997), anti-inflammatory (Alakurtti et al., 2006; Mukherjee et al., 1997) anthelmintic

(Enwerem et al., 2001), antinociceptive (Kinoshita et al., 1998), and anticancer

activities (Fulda and Debatin, 2000; Zuco et al., 2002).

Betulinic acid (1.8) has served as a valuable anti-HIV lead compound and amongst its

derivatives, 3-O-(3',3'-dimethylsuccinyl)betulinic acid (Bevirimat®) (1.9) was extremely

potent. Compound 1.9 inhibited HIV replication with an EC50 < 0.35 nM and TI of 20

000 (Mishra and Tiwari, 2011). Interestingly, 1.9 retained activity even against virus

isolates resistant to NRTIs, NNRTIs, and PIs. Bevirimat® also exhibited synergistic

effects with other AIDS drugs (Lee, 2010).


5

RO H

H

COOH

H

H

1.8 R = H

1.9 R = COOH

O

The two vinca alkaloids, vinblastine (1.10) and vincristine (1.11) were isolated from

Catharanthus roseus (L.) G. Don (Apocynaceae) or Vinca rosea L. (known as “Chang

Chung Hua” in Chinese medicine) (Barnett et al., 1978). Compounds 1.10 and 1.11 are

some of the well-known plant-derived drugs used to treat Hodgkin’s lymphoma and

acute childhood leukemia. Local people of Jamaica and India use C. roseus as a

diuretic, anti-dysenteric, hemorrhagic and antiseptic as well as for the treatment of

diabetes. The anticancer activity of this plant was discovered by serendipity when the

extracts were investigated as a source for potential oral hypoglycemic agents (Cragg

and Newman, 2005). Numerous synthetic analogues of 1.10, e.g. vinorelbine

(Nabelbine®) (1.12) were designed to target other types of tumor or to minimize side

effects shown by compound 1.10. Vinorelbine (1.12) showed activity against non-small

cell lung and advanced breast cancer (Cragg and Newman, 2005; Johnson et al., 1996;

Potier, 1989).

NH

H3CO2CN

N

H3COR

H OAcCO2CH3

OH

H

NOH

1.10 R = CH3

1.11 R = CHO

NH

H3CO2CN

N

H3CO H OAcCO2CH3

OH

H

N

1.12

Another important plant-derived anticancer drug approved for clinical use is paclitaxel

(Taxol®) (1.13). In 1992 paclitaxel was approved for the treatment of ovarian cancer,


6

and later (1994) was approved to treat breast cancer. Paclitaxel (1.13) was first isolated

from the leaves of Taxus brevifolia Nutt. (Taxaceae) (Cragg and Newman, 2005; Wani

et al., 1971). Unfortunately, the supply of paclitaxel (1.13) was limited since only trace

amounts of this compound (0.01% of dry weight of the bark) could be isolated (Cragg

and Newman, 2005). Furthermore, the natural source was nonrenewable as the tree

grows very slowly. As a result, many studies were pursued to synthesize analogues of

1.13 (Denis et al., 1988; Funk and Yost, 1996; Holton et al., 1994). Docetaxel (1.14) is

a clinically used anti-cancer drug which was synthesized from 10-deacetyl-baccatin III

(1.15). Baccatins such as compound 1.15, are key precursors of paclitaxel and are

readily available in various Taxus species (e.g. 1.15 was obtained from T. baccata L.)

(Guenard et al., 1993).

1.13 R1

= COCH3, R2

= HNCOC6H5

R2

H5C6OH

O

O

O

OCOC6H5OH

OR1

OH

H OAc

OH

1.14 R1

= H, R2

= HNCOOC(CH3)3

O

OCOC6H5OH

AcO

OH

H OAc

OH

OH

1.15

In spite of the great success of the pharmaceutical industries in the search for drug lead

compounds from plants, most of the plant biodiversity still remain unexplored (Aremu

et al., 2010; Okem et al., 2012). It is estimated that South Africa has more than 24 000

indigenous plant species and about 3000 of these species are used as medicinal plants.

A large number of these plant species have never been studied. Moreover, 80% of the

South African Black population still relies on traditional medicines for their primary

health care needs. This is because traditional medicine is an important part of the

culture of African people and traditional herbs are generally more accessible and

affordable than the western medicines (Aremu et al., 2010; Okem et al., 2012; Street et

al., 2008).

Traditional medicines are normally assumed to be safe for human consumption as

plants have a long history of usage in the treatment of diseases. However, recent


7

research has shown an increase in the number of deaths due to plant poisoning. Some

factors linked to this acute poisoning include misidentification of plant species and

incorrect preparation or dosage of plant extracts (Fennell et al., 2004; Street et al.,

2008). Hence, the identification, isolation, and evaluation of natural products for their

biological activities and toxicity before they are applied as therapeutic agents are of

great importance.

This project is aimed at the isolation, identification and biological evaluation of natural

products from two Zulu medicinal plants, Elytropappus rhinocerotis (L.f.) Less.

(Asteraceae) and Rhoicissus tridentata (L.f.) Wild & Drumm. subsp. cuneifolia (Eckl.

& Zehr.) N.R. Urton (Vitaceae). The specific aims of the project are listed in Section

1.2. In Section 1.3, a brief description of the organization of this thesis is given.

1.2 Aims of the study

The aims of this study were:

To isolate secondary metabolites from Elytropappus rhinocerotis and to

determine the structures of the isolated compounds.

To develop a method of determining the chemical markers present in E.

rhinocerotis using HPLC-PDA/LCMS and to compare the chemical profiles of

different E. rhinocerotis plants collected from different locations.

To test and compare the uterotonic activity of the methanol (MeOH) extracts of

two Zulu medicinal plants (Rhoicissus tridentata and Gunnera perpensa). These

plants are amongst the most cited plants used in the preparation of Isihlambezo,

a traditional herbal drink taken by pregnant women in their last trimester of

pregnancy to induce labor and improve health of the baby and the mother.

To investigate the phytochemistry of the most uteroactive fraction from R.

tridentata and to evaluate the uterotonic activity of the isolated pure compounds.


8

1.3 Organization of the thesis

Following this chapter (Chapter 1), this thesis contains three more chapters. In Chapter

2, a literature review of the family Asteraceae is presented and the isolation, structural

characterization, and HPLC-PDA analysis of the compounds from E. rhinocerotis are

discussed. Chapter 2 also includes a discussion on the variation of chemical profiles of

different E. rhinocerotis plants collected in different locations. Chapter 3 focuses on the

literature review of oxytocic plants and the isolation and structural characterization of

compounds from R. tridentata. The oxytocic activity of the crude R. tridentata extracts,

G. perpensa, and the compounds isolated from R. tridentata will also be discussed in

Chapter 3. General conclusions about the findings of this research and future

recommendations will be discussed in Chapter 4.

CHAPTER 2: The phytochemistry of Elytropappus rhinocerotis

9


2.1 Introduction

The family Asteraceae includes mainly herbaceous plants, with a rare occurrence of

trees, shrubs, and climbers. To date, Asteraceae is documented as the largest flowering

plant family, with about 25 000 species grouped into 1700 genera and 12 subfamilies

(Funk et al., 2009; Zavada and de Villiers, 2000). These species are widely distributed

throughout the world, but a majority are found in the tropical and subtropical regions,

such as central America, eastern Brazil, the Andes, the Mediterranean, Levant parts of

Middle East, central Asia, South Africa and southwestern China (Bohm and Stuessy,

2001; Jansen and Palmer, 1987; Stuessy, 2010). The species are characterized by the

following features: (i) a group of closely packed flowers into heads (known as an

inflorescence), (ii) small leaf like structures surrounding the flowers (phyllaries), (iii)

the existence of a modified calyx attached to apex of the ovary (pappus) (Barreda et al.,

2012; Jansen and Palmer, 1987; Stuessy, 2010).

The Aims of this Chapter are:

- To present an overview of the documented economic and medicinal uses of

some Asteraceae species, with a particular focus on the South African

indigenous plants.

- To summarise the reported traditional uses, biological activities, and to provide

an overview of the phytochemical studies undertaken for the genus

Elytropappus (an endemic South African Asteraceae genus).

- To present the findings from the phytochemical investigation of Elytropappus

rhinocerotis undertaken in the current study.


10

2.2 Economic and medicinal uses of some Asteraceae plants

Members of the genus Heliathus (sunflower) and Carthamus (safflower) are cultivated

worldwide for their oil and nuts production, as well as the feeding of birds and small

animals (Milner et al., 1945; Torres et al., 2014; Weiss, 2000). Other genera, such as,

Lactuva (lettuce) and Cynara (artichokes) are popular in food production. Several

genera in the Asteraceae family are important in horticulture, for instance, Tagetes and

Chrysanthemums (ornaments) (Jansen and Palmer, 1987), Dahlia (cultigen), Zinnia and

Helenium (garden flower) (Funk et al., 2009).

The orange-yellow carotenoid lutein (2.1) extracted from Tagetes erecta, is well-known

in Europe for providing colour to foods, such as pasta, vegetable oil, margarine,

mayonnaise, confectionery, dairy products, citrus juice and mustard (Hadden et al.,

1999; Piccaglia et al., 1998; Vasudevan et al., 1997). Lutein is approved as a food

colourant in the European Union, Australia and New Zealand, but it is only used in

poultry feed in United States (Otterstätter, 1999; Wrolstad and Culver, 2012).

OH

OH

2.1

The large number of Asteraceae species are found worldwide and their wide array of

natural products make them useful in the treatment of a wide variety of ailments. The

largest genus in this family is the genus Baccharis, which consists of about 500 species.

Baccharis plants are mainly found in the warm temperate and tropical regions of Brazil,

Argentina, Colombia, Chile and Mexico. The genus Baccharis, commonly known as

carqueja, is popularly used in traditional medicine in southern Brazil, Uruguay and

Argentina for the treatment of stomachache, backache, headache and bellyache. The

essential oil composition of several Baccharis species have been studied and slight

variations in the composition is observed. Other types of compounds isolated from this

genus are kaurane, labdane and neo-clerodane diterpenes.


11

Two species from this genus (B. articulate and B. crispa) have been recently cited

amongst the six most popularly used plants for pain relief in Rio Grande do Sul,

Southern Brazil (Florão et al., 2012; Stolz et al., 2014). B. articulate is also taken as a

tonic, febrifuge, diuretic, for digestion, for control of the anemia and weakness,

anthelmintic and weight loss. GC-MS analysis of the volatile oil from B. articulate

from Southern Brazil revealed the major chemical constituents to be β-pinene (39.0%)

(2.2), cis-β-guaiene (9.8%) (2.3), γ-muurolene (5.8%) (2.4), limonene (4.8%) (2.5), α-

pinene (4.5%) (2.6), α-gurjunene (2.7) (4.4%) and spathulenol (4.2%) (2.8) (Simionatto

et al., 2008).

2.2 2.3

H

H

2.4 2.5

2.6

H

2.7 2.8

H

HOH

B. crispa is popularly used in Brazil for the treatment of gastrointestinal, liver and

kidney diseases as well as inflammation. From pre-clinical studies performed on the

crude aerial aqueous extract and butanolic fraction, this plant was shown to possess

antinociceptive and anti-inflammatory properties (Gené et al., 1996; Nogueira et al.,

2011; Paul et al., 2009; Stolz et al., 2014). These activities have been associated with

the presence of a saponin (echinocystic acid, 2.9), rutin (2.10) and other phenolic

compounds in the extracts (de Oliveira et al., 2012; Gené et al., 1996; Stolz et al.,

2014).


12

OHH

OHH O

OHH

2.9

O

OOOH

OH

OHOH

O

O

O

OHOH

OH

OH

OHOH

2.10

Species from the genus Brickella (known as brickellbushes) are native to Mexico and

southwestern United States. In traditional medicine, the herbal tea is prepared from

these species to cure ulcers, migraines, heart diseases and diabetes. The chemical

composition of some Brickella species have been studied, and some isolated

compounds have hypoglycemic and antioxidant properties (Andrade-Cetto and

Heinrich, 2005; Marles and Farnsworth, 1995; Rivero-Cruz et al., 2006). Rivero-Cruz et

al. (2006) studied the phytochemistry of B. veronicaefolia and reported that 86% of its

essential oil consists of benzoates and sesquiterpenoids. A hypoglycemic flavone

(5,7,3'-trihydroxy-3,6,4'-trimethoxyflavone, 2.11) was isolated from the chloroform

extract of the leaves of B. veronicaefolia (Perez G et al., 2000).

OOH

H3COOH O

OCH3

OHOCH3

2.11

Brickella cavanillesii (prodigiosa or hamula) is a bitter-tasting herb widely

commercialized in Mexico (alone or in combination with other plants) for treating

ulcers, dyspepsia, and diabetes. This plant is amongst the 306 most frequently used

species for the treatment of type-II diabetes mellitus (DM), and is sold as a cheaper

alternative to insulin (Andrade-Cetto and Heinrich, 2005; Escandón-Rivera et al., 2012;

Eshiet et al., 2014). Phytochemical studies on the aerial parts led to the isolation of 6-

acetyl-5-hydroxy-2,2-dimethyl-2H-chromene (2.12) (Rodríguez-López et al., 2006),


13

pendulin (2.13), and atanasin (2.14) (Flores and Herrán, 1960; Flores and Herrán, 1958;

Mata et al., 2013). An O-methylated flavonol (brickellin, 2.15) (Iinuma et al., 1985)

was reported as a major constituent of B. cavanillesii and was associated with the

antidiabetic properties shown by this plant (Eshiet et al., 2014). Bioassay-guided

fractionation led to the isolation of several natural products, including, 6-hydroxyacetyl-

5-hydroxy-2,2-dimethyl-2H-chromene (2.16), sesquiterpene lactones (caleins C, 2.17)

and several flavonoids [isorhamnetin (2.18) and quercetin (2.19)]. Compound 2.16-2.19

showed a significant inhibitory activity against the enzyme α-glucosidase.

O

OHO

2.12

OH3CO

H3COOH O

OCH3

O

O

OH

OHOH

OH

2.13

2.14

OH3CO

H3COOH O

OCH3

OCH3

OCH3

OH

2.15

O

OOHOH OCH3

OCH3O

OCH3

O

OHOOH

2.16

HO

OH OH

HO O

O

O

O

2.17

O

OOH

OH

OH

OH

R

R

2.18 OCH32.19 OH

Members of the genus Echinacea are well known in North America and Europe due to

their ability to stimulate the immune system, which is important in the treatment and

prevention of upper respiratory tract infections (Barrett, 2003; Toselli et al., 2009). In


14

traditional medicine, native American Indians use Echinacea species for the treatment

of wounds, burns, insect and snake bites. The roots of these plants are chewed to cure

toothache, throat infection, pain, cough and stomach cramps (Percival, 2000; Shah et

al., 2007). Echinacea extracts and whole plant extracts are commercially prepared as

direct pressed juices, freeze-dried ethanolic or hydrophilic extracts, and powdered dried

leaves and flowers. These products are available in groceries, pharmacies, and health

food stores throughout the world (Barrett, 2003). In the United States alone, Echinacea

products annual sales are estimated to be worth $300 million (Barrett, 2003; Brevoort,

1998).

Artemisia L. is one of the widely used Asteraceae genera across different traditional

medicine systems worldwide. Local communities of India, Myanmar, Pakistan, Nepal,

Bhutan, Afghanistan and Japan uses Artemisia species for fever and eczema, treatment

of wounds and skin diseases, febrifuge, depurative properties, digestive disorders,

epilepsy, psychoneurosis, depression, irritability, insomnia, anxiety, stress, treatment of

amenorrhea and dysmenorrhea. These plants are also used for their anthelminthic,

antiseptic, antispasmodic properties and in ethnoveterinary medicine (Govindaraj et al.,

2008; Rajeshkumar and Hosagoudar, 2012).

The significance of this genus in medicinal chemistry was increased due to the isolation

of an antimalarial drug, artemisinin (1.5) from Artemisia annua (De Vries and Dien,

1996; Rashmi et al., 2014). As discussed in Chapter 1, artemisinin (1.5) was first

isolated from Asian Artemisia specie, A. annua. A. annua is cultivated in Africa and its

tea is well-known for the treatment of malaria (Abad et al., 2012). The only indigenous

member of Artemisia in South Africa is Artemisia afra Jacq. ex Willd (wilde als). A.

afra is amongst the oldest indigenous plants used in the traditional medicine in South

Africa (Van Wyk, 2008a). The local uses of this plant, as well as uses of several South

African Asteraceae plants are discussed below.


15

2.3 Medicinal uses of some South African Asteraceae plants

As mentioned earlier, Asteraceae is the largest family of flowering plants worldwide. In

South Africa these plants often occur in the Cape fynbos biome. Cape fynbos is the

flora of the Western Cape which forms part of the Cape Floral Kingdom and consists of

about 8550 species. Since this fynbos is dominated by Asteraceae, many traditional

medicines used by indigenous people here are derived from plants belonging to this

family (Salie et al., 1996). Several Asteraceae species discussed below are indigenous

to South Africa, with a high occurrence in the Cape fynbos.

Artemisia afra Jacq. ex Willd (wilde als in Afrikaans, lengana in Sotho, and

umhlonyane in Xhosa and Zulu) is a South African indigenous species popularly used

in traditional medicine as a bitter tonic and a stimulant for Cape herbal medicine

(Thring and Weitz, 2006; Van Wyk, 2008b). Amongst other ailments treated by this

plant are respiratory disorders, colic, flatulence, constipation, gastritis, poor appetite,

heartburn, measles, headache, earache, gout, diabetes, malaria, diarrhea and wounds

(Hutchings et al., 1996; Neuwinger, 2000; Van Wyk, 2008a; Watt and Breyer-

Brandwijk, 1962).

Biological studies on A. afra extracts showed that this plant has antimicrobial,

antioxidant, anti-nematodal, antimalarial, cardiovascular, cytotoxic and sedative

properties. A. afra is one of the commercially important medicinal plants in South

Africa, with its first commercial product based on low-thujone material developed as a

tincture (1996) and tablets (2002) under the brand names Healer’s Choice and Phyto

Nova, respectively (Van Wyk, 2011).

Flavonoids found in A. afra extracts include apigenin, chrysoeriol, tamarixetin,

acacetin, genkwanin and kaempferol (Avula et al., 2009; Kraft et al., 2003; Waithaka,

2004). Significant amounts of luteolin and quercetin were isolated from the aqueous

extract (Muganga, 2007; Mukinda et al., 2010; Waithaka, 2004). Luteolin (2.20) and

quercetin (2.19) are reported to be easily extractable, stable under various processing

conditions and selectively quantifiable using HPLC. These compounds were therefore


16

assigned as ideal markers when evaluating A. afra extracts (Markham, 1982; Mjiqiza,

2006; Mjiqiza et al., 2013; Mukinda et al., 2010; Waithaka, 2004).

Several other types of compounds have been isolated from A. afra. These include,

sesquiterpene lactones (for instance, guaianolides and glaucolides) (Jakupovic et al.,

1988), triterpenes (such as, α-amyrin, β-amyrin, and friedelin), as well as alkanes (e.g.

ceryl cerotinate and N-nonacosane) (Silbernagel et al., 1990). Volatile oil from this

plant is very useful and has been used as a substitute for armois oil. Armois oil is

produced by A. vulgarisa L. and is used in perfumes and as a flavoring agent. Although

the composition of this oil varies with geographic origin, some common constituents

have been identified such as 1,8-cineole (2.21), α-thujone (2.22), β-thujone (2.23),

camphor (2.24) and borneol (2.25) (Van Wyk, 2008a, 2011).

OOH

OH O

OHOH

2.20

O

2.21

O

2.22

O

2.23 2.24

O

2.25

OH

Species of the genus Eriocephalus L. are used by South African local communities for

treatment of coughs and colds, flatulence and colic, digestive disorders, as well as

stomach pain. Eriocephalus punctulatus DC. (Cape chamomile) and E. africanus L.

(wild rosemary) were used as diuretic and diaphoretic (Mierendorff et al., 2003). E.

africanus is also used for treating gastro-intestinal, gynaecological complaints,

inflammatory and other dermal complications. The oil extracted from wild rosemary

and Cape chamomile is commercially important in the preparation of perfume, skin care

and beauty products (Amabeoku et al., 2000; Njenga and Viljoen, 2006). The

sesquiterpene, 8-O-isobutanoylcumambrin B (2.26) with antiplasmodial properties was


17

isolated from E. tenuifolius DC. The compound had a low IC50 of 6.25 µg/mL against

Plasmodium falciparum D10, but was unfortunately also cytotoxic (Nthambeleni,

2008).

OH

OH

O

OH

O2.26

Other Asteraceae species commonly cited in South African traditional medicines are

Tarchonanthus camphoratus L. (camphor bush) and Athrixia phylicoides DC. (bush tea

or Zulu tea). The green leaves of the camphor bush are used for a range of ailments

depending on how they are prepared. While the burnt leaves are inhaled to cure blocked

sinuses, asthma and headache (Pretorius, 2008), the infusion of boiled leaves is taken

orally to treat coughs, toothache, abdominal pain and bronchitis. Camphor bush leaves

are also used for massaging the body and as a perfume (Aiyegoro and Van Dyk, 2013;

Amabeoku et al., 2000; Hutchings et al., 1996; Watt and Breyer-Brandwijk, 1962).

The herbal tea prepared from leaves of Athrixia phylicoides is used as a “blood purifier”

for sores and boils. The decoction of leaves and stems is used as a lotion for sore feet,

boils, acne and infected wounds. Leaf infusions are also used as a stimulant, aphrodisiac

drink and gargle for infected throat. In addition, leaf infusions have been reported to

treat hypertension, heart diseases, diabetes, diarrhea and vomiting. Roots are used as a

purgative and to treat coughs (Hutchings et al., 1996; Joubert et al., 2008; Rampedi and

Olivier, 2005; Van Wyk and Gericke, 2000; Watt and Breyer-Brandwijk, 1962).

Phytochemical investigation on the aerial parts of A. phylicoides led to the isolation of

germacren D, linoleic acid and p-hydroxyphenylpropan-3-ol coumarate (Bohlmann and

Zdero, 1977; Joubert et al., 2008). Mashimbye et al. (2006) reported on the isolation of

a new flavonoid, 5-hydroxy-6,7,8,3',4',5'-hexamethoxyflavon-3-ol (2.27) from the

leaves of A. phylicoides (Joubert et al., 2008; Mashimbye et al., 2006). Several

diterpenes related to kaurane, triterpene and thymol derivatives were isolated from the


18

root extracts of some Athrixia species (e.g. Athrixia spp. and A. pinifolia) (Joubert et al.,

2008).

OOCH3

H3CO

H3COOH

OHO

OCH3OCH3

OCH3

2.27

Fouche et al. (2008) conducted a study which was aimed at evaluating the in vitro

anticancer activity of South African plants against breast MCF7, renal TK10 and

melanoma UACC62 human cell lines. For Asteraceae, the hit rate for cytotoxicity

against cancer cell lines was substantially larger than for any of the other plant families.

Schkuhria pinnata (Lam) Kuntze demonstrated anticancer activity and the active

principle was identified as eucannabinolide (2.28). Compound 2.28 showed a total

growth inhibition (TGI) of < 6.25 µg/mL for melanoma UACC62, 7.75 µg/mL for

breast MCF7 and 12.00 µg/mL for renal TK10 cancer cell lines. Eucannabinolide (2.28)

was also isolated from other members of the genus Schkuhria (e.g. S. virgata), where it

exhibited in vivo antileukemic activity (Herz and Govindan, 1980).

2.28

O

O

O OH

H

OH

OH

O

Another Asteraceae representative in the study mentioned above was Oncosiphon

piluliferum. Two active constituents, namely, tetradin A (2.29) and deacetyl-β-

cyclopyrethrosin (2.30) were isolated from the dichloromethane extract of O.

piluliferum. Tetradin A (2.29) exhibited a TGI of 4.50 µg/mL for TK10, 86.10 µg/mL


19

for MCF7 and 18.72 µg/mL for UACC62. Compound 2.30 TGI for TK10 was 5.89

µg/mL, 86.15 µg/mL for MCF7 and 16.92 µg/mL for UACC62 (Fouche et al., 2008).

2.29

OO

OH

OH

2.30

OO

OH

OH

Salie et al. (1996) evaluated the in vitro antimicrobial activity of four indigenous

Asteraceae species (Arctotis auriculata Jacq., Eriocephalus africanus L., Felicia

erigeroides DC. and Helichrysum crispum (L.) D. Don). The organic extract of A.

auriculata and E. africanus showed antimycobacterial activity against Mycobacterium

smegmatis. These findings were very interesting as they showed that these plants also

could inhibit the growth of M. tuberculosis (Salie et al., 1996).

These extracts, as well as extract from F. erigeroides also inhibited growth of

Pseudomonas aeruginosa, a microorganism causing one of the most difficult infections

to treat with normal antibiotics (Levinson and Jawetz, 2002). Organic extracts of E.

africanus and H. crispum and aqueous extract of F. ergeroides exhibited antifungal

activity against Candida albicans. Activity against Staphyllococcus aureus was shown

by organic extracts of A. auriculata and E. africanus (Salie et al., 1996). The

antimicrobial activity of the genus Helichrysum has been extensively studied and other

examples of active species in this genus are mentioned below.

Members of the genus Helichrysum are widely used in Southern African traditional

medicine. Helichrysum is one of the largest genus in the family Asteraceae, consisting

of approximately 500-600 species. About 244-250 of these species occur in South

Africa. Plants from this genus are very popular in traditional medicine for their use in

invoking the goodwill of the ancestors, to induce trances and the aerial parts of several

species that are used for these purposes are commercially available. Examples of these

plants are H. griseum Sond, H. herbaceum (Adrews) Sweet, H. epapposum Bolus and

H. natalitium DC. Helichrysum plants are also used to treat respiratory and gastro-

intestinal disorders, eye conditions, pain and inflammation, menstrual pains,


20

rheumatism and headache. Leaves are often applied as a wound dressing and these

plants are also used to fumigate huts and as bedding to repel insects (Afolayan and

Meyer, 1997; Arnold et al., 2002; Hutchings et al., 1996; Lourens et al., 2008;

Mathekga et al., 2000; Watt and Breyer-Brandwijk, 1962).

Several research groups have extensively studied the antimicrobial activity of

Helichrysum species and most crude extracts usually exhibited higher activity against

Gram-positive organisms than Gram-negative species. Antibacterial compounds, which

are commonly flavonoids, have been isolated from some species. The active compound

isolated from H. aureonitens was galangin (3,5,7-trihydroxyflavone) (2.31).

In a study conducted by Afolayan and Meyer (1997), galangin showed antibacterial

activity against four Gram-positive bacteria (three Bacillus species and Micrococcus

kristinae) and one Gram-negative species (Enterobacter cloaceae). Galangin (2.31) also

exhibited activity against several bacteria and fungi, for instance, six β-lactam-sensitive

and resistant strains of Staphylococcus aureus, sixteen strains of 4-quinolone-resistant

strains of the bacterium, and Aspergillus tamari. In another study, galangin

demonstrated antiviral activity against Herpes simplex virus type 1 and Coxsackie virus

(MIC of 6 µg/mL) (Lourens et al., 2008; Meyer et al., 1997).

OOH

OH OOH

2.31

The active principle from H. odoratissimum was identified as 3-O-methylquercetin.

This compound has antibacterial activity against a wide variety of microorganisms,

including Salmonella typhimurium (Gram-negative, MIC = 50 µg/mL), Staphylococcus

aureus (Gram-positive species, MIC = 6.25 µg/mL) and the fungi (e.g. Candida

albicans, MIC = 12.5 µg/mL) (Van Puyvelde et al., 1989). Pinocembrin chalcone (2.32)

was isolated from H. trilineatum, while pinocembrin (2.33) was obtained as an artefact

during the isolation process from this plant. Both these compounds exhibited anti-

staphylococcal activity (Bremner and Meyer, 1998). Another flavonoid, 5,7-


21

dibenzyloxyflavanone was isolated from H. gymnocomum and it showed activity

against a diverse group of Gram-positive and Gram-negative bacteria as well as a yeast

(Drewes and van Vuuren, 2008; Lourens et al., 2008).

OHOH

OH O

2.32

OOH

OH O

2.33

Interesting antibacterial activity was observed for two chalcones (2.34 and 2.35)

isolated from H. melanacme. 2',4′,6′-trihydroxy-3′-prenylchalcone (2.34) and 1-(3,4-

dihydro-3,5,7-trihydroxy-2,2-dimethyl-2H-1-benzopyran-6-yl)-3-phenyl-(E)-2-propen-

1-one (2.35) inhibited growth of a drug-sensitive H37Rv strain of Mycobacterium

tuberculosis with a MIC of 0.05 mg/mL. The crude extract together with the isolated

chalcones were also evaluated for antiviral activity against the influenza A virus. The

chalcones 2.34 and 2.35 showed lower activity than the crude extract, but the activity

was higher when the chalcones were combined (Lall et al., 2006; Lourens et al., 2008).

OHOH

OH O

2.34

OHO

OH O

OH

2.35

Compounds other than flavonoids with antimicrobial activity have also been isolated

from Helichrysum species. Linoleic and oleic acid were obtained from H. pedunculatum

and they showed antibacterial activity against S. aureus and Micrococcus kristinae

(MIC = 1.0 mg/mL) (Dilika et al., 2000). The active compound (kaurenoic acid) from

H. kraussii inhibited growth of E. coli, Bacillus cereus, B. subtilis, S. aureus and

Serratia marcescens. A prenylated butyrylphloroglucinol (2.36) (MIC = 100 µg/mL)

was also isolated from H. kraussii and it showed activity against similar bacteria as


22

kaurenoic acid (2.37) as well as B. pumilis and Micrococcus kristinae (Bremner and

Meyer, 2000; Lourens et al., 2008)

2.36

OH

OH

OH

O COOH

HH

2.37

From the crude extract of H. caespititium, two phloroglucinols (caespitin 2.38 and 2.39)

with antimicrobial activity were isolated. Caespitin (2.38) inhibited growth of S. aureus,

Streptococcus pygenes, Cryptococcus neoformans, Trichophyton rubrum, Trichophyton

mentagrophytes and Microsporum canis (Mathekga et al., 2000). 2-Methyl-4-[2′,4′,6′-

trihydroxy-3′-(2-methylpropanoyl)-phenyl]but-2-enyl acetate (caespitate) (2.39) was

active against several microorganisms, including Bacillus cereus, B. pumilis, B.

substilis, Microsporum kristinae and S. aureus (Mathekga et al., 2000). At a

concentration of 0.5-1.0 µg/mL, the antifungal activity of caespitate (2.39) was

observed against Aspergillus flavus, A. niger, Cladosporium cladosporioides, C.

cucumerinum, C. sphaerospermum and Phytophtora capsici (Lourens et al., 2008;

Mathekga et al., 2000)

2.38

OH

OH OH

O

2.39

OH

OH OH

O

OO

In summary, Asteraceae plants are used extensively in ethnomedicines worldwide and

many of the uses are associated with the treatment of infectious diseases, for instance,

many genera are used to treat respiratory disorders and wounds. The chemical

compositions of many genera in the family have been studied, and a wide variety of

compounds have been isolated. This is not surprising since this family has a large


23

morphological diversity of genera. The most common groups of compounds identified

in this family include flavonoids, sesquiterpenes, diterpenes and acylated

phloroglucinols. The biological/ pharmacological activities of the plant extracts and the

compounds isolated from Asteraceae species have up to now not received enough

attention. In this chapter, we intend to report on the isolation and structural elucidations

of the compounds isolated from Elytropappus rhinocerotis.

2.4 The genus Elytropappus

2.4.1 Introduction

The genus Elytropappus Cass. (Asteraceae, tribe Gnaphaliea) derives its name from the

Greek words elytron (sheath) and pappos (fluff) which is appropriate considering the

fluffy feathery appearance of the top part of the seeds displayed by several species in

this genus. This genus was first described by Cassini in 1816 based on Gnaphalium

hispidum (recently known as Elytropappus hispidus). A comprehensive taxonomical

study on Elytropappus was later carried out by Levyn in 1935. Her findings led to the

grouping of the eight Elytropappus species into three groups (Group 1: E. cyathiformis

and E. hispidus; Group 2: E. longifolius, E. gnaphaloids, E. scaber and E. glandulosus;

Group 3: E. rhinocerotis and E. adpressus) (Levyns, 1926, 1935).

Koekemoer re-assessed the taxonomy of Elytropappus to establish the rank of its formal

and informal grouping. In this study Koekemoer divided Elytropappus into three

genera. The species which were previously in Group 2, together with Stoebe intricata,

were grouped under a new genus Mytovernic, and Group 3 species (E. rhinocerotis and

E. adpressus) were grouped under a new genus Dicerothamnus (Koekemoer, 2002).

However, the proposed reclassification has not yet been published in a scientific

journal; therefore, we will refer to Elytropappus in this thesis as a single genus.


24

All Elytropappus species are endemic to South Africa, particularly they are common in

Cederberg (part of Cape floristic region). With the exception of E. rhinocerotis,

Elytropappus species are not well known and there is no literature about their medicinal

or ecological uses. E. rhinocerotis (Fig. 2.1), commonly known as “renosterbos or

rhinoceros bush” is a bush-shrub of 1-2 m in height with small grayish-green leaves and

tiny flower heads. During the shedding of the seeds, the brown chaffy bracts around

each flower head open up, giving the plant a brownish fluffy appearance (Dorchin and

Gullan, 2007; Levyns, 1935; Pool et al., 2009).

Figure 2.1: Habitat and aerial parts of E. rhinocerotis

(Photo: http://botany.cz/cs/dicerothamnus-rhinocerotis/)

“Renosterbos” is a dominant plant in Renosterveld. This vegetation, which is believed

to have derived its name from “Renosterbos”, was first described by Sparrman (a

distinguished South African traveler) in 1775. “Renosterbos” is also found as far north

as the Namaqualand and Richtersveld, the great escarpment around Molteno, and it also

extends to the southern parts of Eastern Cape to East London. This plant tolerates both

snow and fire and is abundant in heavily grazed areas as it is unpalatable to livestock

(Proksch et al., 1982). As a result, farmers consider “renosterbos” as a major weed and

a lot of research has been done on the eradication strategies and its biocontrol

(Koekemoer, 2002).


25

2.4.2 Traditional uses

In traditional medicine, the powdered young tips and branches of E. rhinocerotis are

used to treat colic, wind and diarrhoea in children. Adults take the infusions of the twigs

(in brandy or wine) to treat indigestion, dyspepsia, gastric ulcers and stomach cancer.

These infusions are also used as a tonic drink to improve appetite. E. rhinocerotis

became popular for its use in the treatment of influenza and fever in the flu epidemic of

1918 (Hutchings et al., 1996; Thring and Weitz, 2006; Watt and Breyer-Brandwijk,

1962).

2.4.3 Phytochemistry and biological activities

Dekker et al. (1988) isolated a new labdane diterpene, rhinocerotinoic acid (2.40) from

the aerial parts of E. rhinocerotis. This compound exhibited anti-inflammatory activity

in both non-adrenalectomised and adrenalectomised rats. Gray et al. (2003) tried to

isolate rhinocerotinoic acid from E. rhinocerotis but could not obtain this compound.

Studies on the chemical composition of the leaf resin indicated that the methoxylated

flavones, cirsimaritin (2.41), hispidulin (2.42), eupafolin (2.43) and quercetin (2.44)

were the major products (Proksch et al., 1982).

Benzoic acid (2.45), its derivatives [hydroxybenzoic acid (2.46), protocatechuic acid

(2.47) and veratric acid (2.48)], as well as cinnamic acid derivatives [p-coumaric acid

(2.49), ferulic acid (2.50) and sinapic acid (2.51)] were reported as minor products from

the leaf resin of E. rhinocerotis (Proksch et al., 1982). However, it is unclear whether

compounds 2.41-2.48 are produced by E. rhinocerotis or by the insects that use this

plant as a habitat. To date, three species of gall-inducing Diptera (Spathulina peringueyi

Bezzi and two species belonging to the Cecidomyiidae family) are reported to reside

within E. rhinocerotis (Dorchin and Gullan, 2007). Cardiac glycosides, saponins and

tannins were detected from the crude sample of E. rhinocerotis (Scott et al., 2004).


26

However, these observations were based on colour reactions and the results should be

treated with care.

COOH

O

2.40

O

OH

OHH3CO

OH

O2.41

O

OH

OHH3CO

H3CO

O2.42

O

OH

OHH3CO

OH

O

OH

2.43

O

OH

OH

OH

OOH

OH

2.44

COOH

R1

R2

2.45 R1 = H, R

2 = H

2.46 R1 = OH, R

2 = H

2.47 R1 = OH, R

2 = OH

2.48 R1 = OCH3, R

2 = OCH3

OH

COOH

R1

R2

2.49 R1 = H, R

2 = H

2.50 R1 = OCH3, R

2 = H

2.51 R1 = OCH3, R

2 = OCH3

Knowles (2005) reported that the extracts of E. rhinocerotis showed antifungal

properties against Botrytis cinerea, a fungal pathogen causing grey mould rot on a large

number of economically and horticulturally important crops. This activity was more

effective when the extracts were combined with synthetic fungicides. The methanol

extract of the aerial parts showed moderate antimicrobial activity against S. aureus. A

zone inhibition of 13 mm was observed compared to 9 mm (no activity) and 27 mm for

ciprofloxacin (control) (Knowles, 2005).

The above discussion demonstrates that the phytochemistry of E. rhinocerotis is not

well understood. Even though some compounds were reported to occur in this plant,

further studies are required to confirm their occurrence and to isolate new compounds.


27

Herewith we report further phytochemical investigation of the aerial parts of E.

rhinocerotis. The biological activities of the isolated compounds will also be evaluated.

We will also report on the variation in the chemical composition of E. rhinocerotis

plants collected in different locations.

2.5 Results and discussion

2.5.1 Introduction

The leaves and branches of E. rhinocerotis were collected on farm Weltevreded in

Sneeuberg, Murraysburg, Western Cape. After air drying and milling, the aerial plant

material was extracted with dichloromethane (DCM) - methanol (MeOH) (1:1). This

extract was fractionated by silica gel chromatography to afford 6,7-dimethoxycoumarin

(2.52), 5,7,4'-trihydroxyflavone (2.53), 5,7-dihydroxy-4'-methoxyflavone (2.54), 5,7-

dihydroxy-6,4'-dimethoxyflavone (2.55), kaempferol 3-methyl ether (2.56), (+)-13-epi-

labdanolic acid (2.57), (+)-labdanolic acid (2.62), (+)-labdanolic acid methyl ester

(2.64) and (+)-labdanediol (2.66). Although other compounds have been isolated from

E. rhinocerotis (Proksch et al., 1982), the isolation of compounds 2.52-2.57, 2.62, 2.64

and 2.66 from this species has not yet been reported. In the next section, the structural

determination of these compounds is discussed.

2.5.2 6,7-Dimethoxycoumarin (2.52) O

H3CO

H3CO O

2.52

2

45

78a

4a

Compound 2.52 was fluorescing violet on TLC under UV light and showed a strong

UV absorption peaks at λmax 228 and 343 nm (Fig. 2.2). These observations suggested

the presence of a coumarin chromophore in compound 2.52 (Hammoda et al., 2008;

Steck and Bailey, 1969). The structure of 2.52 was further characterised by MS and


28

NMR data. A pseudo-molecular ion peak at m/z 229.0482 [M+Na]+, which is in

agreement with a molecular formula of C11H10O4, was observed in the HRMS spectrum.

Figure 2.2: UV/Vis absorption spectrum of compound 2.52

The appearance of two singlets integrating for three protons each at δH 3.91 and 3.94 in

the 1H NMR spectrum (Plate 1a) indicated the presence of two methoxy groups. The

aromatic protons appearing as singlets at δH 6.83 (H-5) and 6.85 (H-8) are para to each

other, and this suggested that the methoxy groups on the aromatic ring are in ortho

positions (C-6 and C-7). The two doublets observed at δH 6.28 and 7.61 in 1H NMR

spectrum were assigned to H-3 (J = 9.5 Hz) and H-4 (J = 9.5 Hz) respectively. In the 13C NMR spectrum (Plate 1b), ten signals corresponding to eleven carbons (the two

OCH3 carbon signals have the same chemical shift) were observed.

The DEPT NMR spectrum (Plate 1d) displayed six protonated carbons. The upfield

signal at δC 56.4 in the 13C and DEPT NMR spectra was assigned to the two methoxy

carbons. The remaining four protonated carbon signals in the DEPT NMR spectrum

were assigned to the methine carbons (C-3, C-4, C-5, and C-8). Four oxygen-linked

non-protonated carbons were observed at δC 161.4 (C, C-2), 152.9 (C, C-7), 150.1 (C,

C-8a) and 146.4 (C, C-6). The 1H, 13C NMR and the mass spectroscopy data led to the

assignment of compound 2.52 as 6,7-dimethoxycoumarin.


29

Further structural confirmation was achieved by analysing the HMBC spectrum (Plate

1f). Correlations were observed between the aromatic singlets (H-5 and H-8) and the

methoxy bearing carbons (C-6 and C-7) (Plate 1f and Fig. 2.3). A correlation between

H-4 and the carbonyl carbon (C-2) also confirmed the proposed structural connection

(Fig. 2.3). The experimental NMR data of compound 2.52 was in agreement with

literature data for 6,7-dimethoxycoumarin, commonly known as scoparone (Céspedes et

al., 2006).

O

H3CO

H3CO O

H

H

25

7 8a

4a

Figure 2.3: Key HMBC correlations in compound 2.52

Scoparone (2.52) was previously isolated from several species in the Asteraceae family,

such as Artemisia tridentate (Imamura et al., 1977), Haplopappus foliosus (Urzua,

2004) and Artemisia capillaris (Jang et al., 2006). Coumarins (including 6,7-

dimethoxycoumarin) have been reported to exhibit antibacterial and antifungal activity

against S. aureus, S. agalactiae, S. uberis, S. dysgalactiae, E. coli and Salmonella

(Céspedes et al., 2006). Other activities of scoparone (2.52) include vasodilation,

immunosuppression, radio-protection and anticoagulation activity (Gakuba, 2010).


30

2.5.3 5,7,4'-Trihydroxyflavone (2.53)

O

OOH

OH

OH

2.53

4'2'

6'2

46

8

Compound 2.53 appeared as a brown spot on TLC under UV light and showed up as a

bright yellow spot after treating the TLC with p-anisaldehyde/ H2SO4 stain followed by

heating. The UV absorption spectrum (Fig. 2.4) of this compound showed λmax at 267

and 338 nm, which is in agreement with the reported values for flavones (Rijke et al.,

2006). A pseudo-molecular ion peak at m/z 269.0454 [M-H]- observed in the HRMS

spectrum is in agreement with a molecular formula of C15H10O5.


In the 1H NMR spectrum (Plate 2a) of compound 2.53, two meta-coupled doublets were

observed at δH 6.18 (1H, d, J = 2.0 Hz, H-6) and 6.42 (1H, d, J = 2.0 Hz, H-8). This

indicated the presence of a tetra-substituted A-ring of a flavone. A deshielded singlet

appearing at δH 6.56 was assigned to H-3 of ring C. Two ortho-coupled signals, each

integrating to two protons was observed at δH 6.92 (2H, d, J = 8.8 Hz, H-3', 5') and 7.84

(2H, d, J = 8.8 Hz, H-2', 6'), suggesting that the B-ring of the flavone was di-substituted

at para positions.


31

In the 13C NMR spectrum (Plate 2b), 15 signals were observed. The DEPT 135

spectrum (Plate 2d) revealed that the structure contained 7 methine carbons (of which

two were overlapping) and 8 non-protonated carbons. A deshielded carbon signal at δC

183.7 was assigned to the carbonyl carbon (C-4). The other oxygen-linked carbons

appeared at δC 166.1 (C, C-5), 165.8 (C, C-2), 162.9 (C, C-4', C-8a) and 159.6 (C, C-7).

In the HMBC spectrum (Plate 2f), correlations were observed between H-3, C-2, C-4,

C-1', C-4a and C-8a; H-6, C-5, C-7 and C-4a; H-2'/6', C-2, C-1' and C-4'; as well as

between H-3'/5', C-4' and C-2. Some of these correlations are shown in Figure 2.5.

Based on the spectral information obtained from NMR, mass, UV/Vis absorption

spectra and comparison with literature values (Ersoz et al., 2002), compound 2.53 was

assigned as 4',5,7-trihydroxyflavone, also known as apigenin.

O

OOH

OH

OH

HH

H

H

4'

6'2

4

8

Figure 2.5: Key HMBC correlations in compound 2.53

Asteraceae plants are well-known for producing a wealth of flavonoids. These plants

have been reported to produce nearly all types of known flavonoids. Amongst the

flavonoids featuring a six-membered C-ring, flavanones, flavones and flavonols are the

most common groups. The most widely occurring substitution pattern displayed by

flavones in this family is 5,7,4'-trioxygenation (apigenin type) and 5,7,3',4'-

tetraoxygenation (luteolin type). The occurrence of apigenin (2.53) and its glycosidic

derivatives have been reported in a large number of Asteraceae species and was

identified in at least one species in 118 genera out of over 430 genera in this family

(Bohm and Stuessy, 2001).


32

Some genera that are reported to contain of apigenin (2.53) belong to the same tribe

(Gnaphalieae) as Elytropappus. Example of these genera are Cassina, Ozothamnus,

Odixia, and Gnaphalium (Wollenweber et al., 2005; Wollenweber et al., 1997b;

Wollenweber et al., 2008; Zheng et al., 2013). As mentioned earlier, this is the first

report on the occurrence of apigenin (2.53) in E. rhinocerotis. Previously isolated

flavones (2.41-2.43) from this plant displayed 5,6,7,4'-tetraoxygenation substitution

pattern (scutellarein type), which is slightly different to the apigenin substitution (5,7,4'-

trioxygenation) (Bohm and Stuessy, 2001).

Flavonoids have a potential as chemoprevention and chemotherapeutic agents. The

inhibition of malignant human cancer cells was shown to be through mechanisms such

as cell cycle arrest, induction of apoptosis, reversal of multi-drug resistance,

antiproliferation, antioxidant, inhibition of angiogenesis and inhibition of telomerase

activity (Lindenmeyer et al., 2001; Ramos, 2007; Ren et al., 2003). Apigenin was also

reported to regulate diabetes mellitus, thyroid dysfunction and lipid peroxidation (Panda

and Kar, 2007).

2.5.4 5,7-Dihydroxy-4'-methoxyflavone (2.54)

O

OOH

OH

OCH3

2.54

Similarly to compound 2.53, compound 2.54 stained bright yellow on TLC treated with

p-anisaldehyde/ H2SO4 stain followed by heating. This was indicative of the presence of

a flavonoid moiety in this compound. The UV absorption spectrum (Fig. 2.6) supported

the presence of a flavone structure as it showed absorption peaks at λmax 268 and 333

nm (Rijke et al., 2006). In the HRMS spectrum, a pseudo-molecular ion peak at m/z

283.0609 [M-H]-, in agreement with the molecular formula of C16H12O5, was observed.


33


The 1H (Plate 3a) and 13C (Plate 3b) NMR spectra of compound 2.54 were similar to

those of compound 2.53. The only difference between these spectra was the presence of

the methoxy singlet at δH 3.92 and δC 55.1 in the 1H (Plate 3a) and 13C (Plate 3b) NMR

spectra of compound 2.54. The position of this methoxy group was determined by

analysing the HMBC NMR spectrum (Plate 3f). A HMBC correlation (Fig. 2.7) was

observed between the methoxy proton signal and C-4' (δC 162.8), which suggested that

the methoxy was attached to C-4'. The structure of compound 2.54 was assigned as 5,7-

dihydroxy-4'-methoxyflavone (also known as acacetin or 4-O-methyl apigenin) and the

experimental NMR data was in agreement with the reported data for this compound

(Soon-Ho et al., 2003).

O

OOH

OH

OCH3

HH

H75 4

2

4'2'

Figure 2.7: Some HMBC correlations in compound 2.54


34

The isolation of acacetin (2.54) is reported for first time from E. rhinocerotis but this

compound is common in other Asteraceae plants. Acacetin (2.54) was isolated from

Blainvillea rhomboidea (Gomes et al., 2010), Centaurea furfuracea (Fakhfakh et al.,

2005), Microglossa pyrifolia (Kohler et al., 2002), some species in the genus Arnica

(Merfort, 1984; Schmidt and Willuhn, 2000), several Artemisia species (Valant-

Vetschera and Wollenweber, 1995) and some Baccharis species (Wollenweber et al.,

1986a). 2.54 were also isolated from a Korean medicinal plant, Dendranthema

zawadskii var. latilobum Kitamura, where it showed antimicrobial activity against

Candida species with an inhibition zone of 9-12 mm. This compound also showed

moderate anticancer activity against human lung carcinoma (A549), skin melanoma

(B16F1) and mouse melanoma (SK-MI-2) with an IC50 of > 40 µg/mL (Rahman and

Moon, 2007).

2.5.5 5,7-Dihydroxy-6,4'-dimethoxyflavone (2.55)

O

OOH

OH

OCH3

H3CO

2.55

Compound 2.55 was identified as a flavone based on the colour of its spot on TLC and

the UV absorption spectrum pattern. The compound stained yellow on TLC treated with

p-anisaldehyde stain and the λmax at 274 and 333 nm was obtained from the UV

spectrum (Fig. 2.8). A pseudo-molecular ion peak at m/z 313.0714 [M-H]- observed in

the HRMS spectrum is in agreement with the molecular formula (C17H14O6).


35


Two methoxy signals were observed in the 1H and 13C NMR spectra (Plate 4a & Plate

4b) at δH 3.89/ δC 55.5 and 4.04/ δC 60.8. The correlation between the methoxy protons

at δH 3.89 and the C-4' carbon in the HMBC spectrum (Plate 4f) confirmed that the

methoxy is attached to C-4'. The HMBC spectrum also showed that the second methoxy

was attached to C-6. The 6-OMe experiences steric hindrance from the two ortho

substituents which affects the aryl-O-bond and causes the methoxy group to adopt an

out-of-plane conformation. This conformation results in an inefficient electron

conjugation between the lone-pair of the methoxy oxygen and the aromatic ring which

decreases electron density at the methoxy group and the ring carbons in the ortho and

para positions to the methoxy group (Agrawal, 1989). This explains the observed

downfield shift of the 6-OMe and C-5 signals in the 1H and 13C NMR spectra.

The combination of 1H, 13C NMR, UV/Vis absorption and mass spectral data led to the

assignment of compound 2.55 as 5,7-dihydroxy-6,4'-dimethoxyflavone. The proposed

structure was further confirmed by HMBC correlations observed between 5-OH signal,

the methoxy bearing carbon (C-6) and C-4a; H-3, C-2 and C-1' and between H-8, C-7

and C-6 (Plate 4f, Fig. 2.9).


36

O

OOH

OH

OCH3

HH3COH

H 4'6'

24

8

2'

Figure 2.9: Important HMBC correlations in compound 2.55

5,7-Dihydroxy-6,4'-dimethoxyflavone (2.55), commonly known as pectolinarigenin, is

isolated for the first time from E. rhinocerotis. The substitution pattern (5,6,7,4') shown

by this compound is similar to the pattern displayed by flavones 2.41-2.43 which were

previously isolated from E. rhinocerotis (Proksch et al., 1982). Pectolinarigenin (2.55)

was isolated from several Asteraceae species, including some members of the genus

Artemisia (Valant-Vetschera and Wollenweber, 1995), Erigeron breviscapus (Vant.)

Hand.-Mazz. (Zhang et al., 2000), Heterotheca latifolia (Rojo et al., 2004), Arnica

species (Merfort, 1984; Schmidt and Willuhn, 2000), Tanacetum macrophyllum Willd

(Ivancheva and Stancheva, 1997), Baccharis species and Brickellia californica (Torrey

& Gray) A. (Wollenweber et al., 1997a; Wollenweber et al., 1986b).

Watanabe et al. (2014) reported on the isolation and phytotoxicity of pectolinarigenin

(2.55) from Onopordum acanthium L. (Watanabe et al., 2014). 2.55 was also isolated

from Cirsium chanroenicum and it showed in vitro and in vivo anti-inflammatory and

anti-analgesic activities (Lim et al., 2008). Pectolinarigenin (2.55) prevented hepatic

injury induced by D-galactosamine (GalN) in rats (Yoo et al., 2008).


37

2.5.6 Kaempferol 3-methyl ether (2.56)

O

OOH

OH

OH

OCH3

2.56

Compound 2.56 showed a yellow spot on TLC which intensified to a brown colour after

spraying the TLC with anisaldehyde in concentrated H2SO4. This compound showed

strong UV absorption peaks at λmax 266 and 350 nm, which is in agreement with the

reported values for flavonols (Fig. 2.10) (Moiseev et al., 2011). A pseudo-molecular ion

peak at m/z 299.0721 [M-H]- was obtained from the LRMS spectrum. Two ortho-

coupled doublets (integrating to 2H each) were observed in 1H NMR spectrum (Plate

5a) at δH 6.95 and 8.00. These indicated the presence of an AA'XX' system of a 1,4

disubstituted aromatic ring. Two more doublets (integrating to 1 proton each) were

observed at δH 6.23 (J = 2.0 Hz) and 6.44 (J = 2.0 Hz). These were meta-coupled and

were assigned to the two protons of tetra-substituted A-ring of a flavone.


A methoxy signal appeared at δH 3.79/ δC 60.6 in 1H and 13C NMR spectrum (Plate 5b).

This signal is in a deshielded position when compared to the methoxy of 5,7-dihydroxy-

4'-methoxyflavone (2.54). This deshielding suggested that the methoxy group is in a

sterically congested environment, which disturbs conjugation between the methoxy

oxygen lone pairs and the C-ring. As a result, the methoxy group is in a less electron

dense environment and therefore deshielded. An HMBC correlation (Plate 5f) was

observed between the methoxy protons and carbon signal at δC 138.1. This carbon was


38

assigned as C-3 since no proton showed a cross peak to this carbon in the HSQC

spectrum (Plate 5e). Also, C-3 is more deshielded in this compound when compared to

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